CN116267604A - Method for obtaining acer truncatum aseptic seedlings and directly inducing aseptic buds - Google Patents
Method for obtaining acer truncatum aseptic seedlings and directly inducing aseptic buds Download PDFInfo
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- CN116267604A CN116267604A CN202310135046.3A CN202310135046A CN116267604A CN 116267604 A CN116267604 A CN 116267604A CN 202310135046 A CN202310135046 A CN 202310135046A CN 116267604 A CN116267604 A CN 116267604A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
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- Developmental Biology & Embryology (AREA)
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Abstract
The invention provides a method for obtaining aseptic seedlings from acer truncatum seed embryos and directly inducing axillary buds by aseptic seedling with bud stem sections, which comprises the steps of disinfection of explants, germination culture, subculture and axillary bud induction culture. The invention has the advantages of low pollution rate of explants, high propagation speed and short cultivation period, not only provides a way for rapid propagation of excellent sterile seedlings, but also provides possibility for establishment of industrial seedling and genetic transformation systems.
Description
Technical Field
The invention belongs to the field of tissue culture of Acer truncatum, and in particular relates to a method for sterilizing an embryo explant of Acer truncatum and a method for directly inducing rapid proliferation of aseptic buds by aseptic seedling with bud stem segments.
Background
Acer truncatum (Acer truncatum Bunge) belongs to the deciduous small arbor of Acer (Acer) of Aceraceae, and is named as North China Acer truncatum Ping Jiqi, which is a special tree species in China, and is named as ancient gold ingot Acer truncatum of China. The height of the tree is 8-20 m, and the tree is mainly distributed in yellow river basin of China. Acer truncatum Bunge is used as a special economic tree species in China, and is determined to be an excellent woody oil tree species due to high kernel oil content, strong ecological adaptability and high economic value; the seed oil contains the specific substance which can promote the repair and regeneration of damaged nerve tissues, namely the nervonic acid, and the No. 9 file of the Ministry of health of the people's republic of China is approved in the year 2011 at the month of 3 and 22, so that the acer truncatum seed oil is approved as a new resource food, and enters into edible vegetable oil lines in China formally, and the development of the acer truncatum industry is further promoted. Acer truncatum was identified as a precious woody oil tree species by the national forestry and grassland bureau in 2017. At present, few reports are about Acer truncatum tissue culture, and the Acer truncatum tissue culture is still in a starting stage. The method mainly obtains the tissue culture seedling of the acer truncatum from a series of processes of disinfection, germination culture, subculture, axillary bud induction culture and the like of the acer truncatum explant, greatly shortens the breeding period of the acer truncatum, has simple operation, good growth vigor of sterile bud seedlings and low pollution rate, and provides possibility for industrial seedling culture and transgenic technology of the acer truncatum.
At present, tissue culture technical researches of Acer truncatum have been reported as follows:
tissue culture method of 201210221749.X Acer truncatum
202010729421.3 method for quickly seeding Acer truncatum
2020110208208. X method for promoting germination of Acer truncatum seeds
Compared with the above patent, the invention has the advantages of simple operation, short culture period, simple formula of culture medium, low cost, and is especially suitable for rapid propagation of Acer truncatum.
Disclosure of Invention
The invention aims at solving the problem that the sterilization of the prior Acer truncatum seeds is not thorough, and provides a sterilization method. The disinfection method is simple and efficient, has good disinfection effect, and lays a solid foundation for the establishment of a tissue culture rapid propagation system of Acer truncatum.
A method for disinfecting acer truncatum seeds, comprising the following steps:
(1) Explant collection: mature, full and pest-free acer truncatum seeds
(2) Explant treatment: and (5) placing the collected Acer truncatum seeds in a refrigerator at the temperature of 4 ℃ for refrigeration, and taking out after 1 d. Adding a proper amount of washing powder, placing the washing powder under tap water for washing for 20-50 min, peeling off exodermis, continuing washing for 1h, and finally washing with sterile water for 3-5 times.
(3) Explant sterilization: placing the explant washed by sterile water in an ultra-clean workbench, soaking the explant in 70% alcohol solution for 30-60 s, and washing 3 times by sterile water. Soaking Acer truncatum seeds in 0.1% mercuric chloride solution for 3-10 min (the specific time depends on the lignification degree of seed coats), and repeatedly cleaning with sterile water for 5-8 times. Finally, the seeds are placed on sterile filter paper to absorb redundant moisture on the surfaces of the seeds.
The effect of disinfecting the acer truncatum seeds by only adopting one disinfectant is not ideal, the explant material is treated by alcohol with the volume concentration of 70 percent and 0.1 percent mercuric chloride together, and the disinfectant remained on the surfaces of the seeds is cleaned for many times by sterile water, so that the ideal disinfecting and sterilizing effect can be achieved. And the inner seed coats of the Acer truncatum seeds are reserved when the Acer truncatum seeds are sterilized, otherwise, the vigor of the Acer truncatum seeds is greatly reduced, and the germination of the Acer truncatum seeds is difficult.
The second purpose of the invention is to provide a method for directly inducing the young and tender bud stem explants of Acer truncatum to generate cluster buds, and the method directly induces axillary buds through the bud stem explants, so that the induction rate is high, the growth condition is good, and a foundation is laid for industrial seedling culture and later transgenic technology of Acer truncatum.
A method for directly inducing rapid proliferation of aseptic buds of acer truncatum seedlings with bud stem segments comprises the following steps:
axillary bud induction culture: the aseptic seedling of Acer truncatum Bunge is placed in an ultra-clean workbench, and leaves are cut off, leaving 1cm long leaf stalks. Cutting the plant into stem segments with at least one axillary bud and 1-3 cm long. Inoculating the stem section of Acer truncatum with single bud into the culture medium of MS+1.0-5.0 mg/L6-BA+0.1-0.5 mg/L IBA for axillary bud induction culture. Culturing under dark culture and light condition.
In the method, the culture temperature is 24 ℃ and 2 ℃, the illumination culture intensity is 2100-2200lx, and the illumination culture time is 12-15h/d.
The culture medium for axillary bud induction in the above method is preferably: MS+3.0 mg/L6-BA+0.1 mg/L IBA.
The culture medium used in the method is added with 30g/L of sucrose and 7g/L of agar, and the pH is adjusted to 5.5-5.8.
In addition, the research on tissue culture of Acer truncatum is relatively rare. The differences and advantages of the present invention are summarized as follows:
1. the invention successfully breeds the aseptic seedlings by utilizing the acer truncatum seed embryo, and the complete aseptic seedlings can be obtained only by 30 days. Thereby shortening the seedling raising period, saving seedling raising materials, solving the problems of more materials, long period and the like required by the seedling raising in the traditional seedling raising method, greatly reducing the production cost and improving the production efficiency. The aseptic seedling has the characteristics of short growth period, more branches, high rooting rate, more root systems and strong growth of tissue culture seedlings.
2. The invention directly adopts the stem segment of the aseptic seedling when axillary bud induction is carried out, thereby ensuring the activity of the stem segment with buds, avoiding the pollution of the explant caused by external secondary sampling, saving time and avoiding the waste of the explant material.
3. The invention directly induces the aseptic bud by the stem section of the acer truncatum bunge with bud, which remarkably saves the acquisition time of the aseptic bud, and the obtained aseptic bud is robust and active, thus forming a set of rapid propagation technical system suitable for the tissue culture early stage of the aseptic bud of the acer truncatum bunge tissue culture seedling, and laying a foundation for the industrial seedling culture and the later genetic engineering improvement of the acer truncatum bunge.
4. The invention adopts the traditional formula to carry out germination culture on Acer truncatum seeds in the early stage, discovers that the sterile seedlings have long potential difference, fewer branches and fewer leaves, and is not beneficial to the subsequent proliferation culture. In the aspect of axillary bud induction culture, the original formula is utilized to find that the axillary bud growth condition is poor, and the axillary bud is unfavorable for being used as the subsequent generation, rooting and seedling hardening transplanting. After the formula of the culture medium is regulated, the branch count and growth vigor of the tissue culture seedling of Acer truncatum are greatly improved.
5. The invention obtains a formula suitable for germination of Acer truncatum seed embryo and induction of axillary buds with bud stem segments through a large number of experiments, greatly improves the differentiation rate of the axillary buds and obtains the tissue culture seedling of the Acer truncatum with strong growth.
6. When axillary buds are induced, the stem segments with buds are not vertically inserted into the culture medium by adopting a traditional method, but are spread on the culture medium, so that the contact surface area of the stem segments and the culture medium is greatly increased, and the transportation of nutrient substances and moisture is facilitated.
Drawings
FIG. 1 shows different treatment modes of inner and outer seed coats of the acer truncatum seed embryo in the process of sterilizing;
FIG. 2 shows the inoculation of Acer truncatum seed embryo containing low concentration GA 3 15d after MS medium;
FIG. 3 is a photograph of a seed embryo of Acer truncatum of the present invention after being inoculated into a MS medium containing 2,4-D for 7D;
FIG. 4 is a photograph of a seed embryo of Acer truncatum of the present invention after being inoculated into a MS medium containing 2,4-D for 15D;
FIG. 5 is a photograph of a seed embryo of Acer truncatum of the present invention after being inoculated into a MS medium containing 2,4-D for 30D;
FIG. 6 shows the inoculation of Acer truncatum seed embryo containing high concentration GA 3 A photograph after 30d of MS medium;
FIG. 7 is a photograph showing the roots of the embryo of Acer truncatum seeds of the present invention inoculated into MS medium containing 2,4-D for 30D;
FIG. 8 is a photograph of a acer truncatum seed embryo of the present invention after 35d of aseptic seedling subculture;
FIGS. 9, 10 and 11 are photographs of the roots of the sterile seedlings of the Acer truncatum seed embryo of the present invention after 15d, 30d and 60 d;
FIG. 12 is a photograph of a Acer truncatum with bud stem section of the present invention after 18d cultivation in an axillary bud induction medium;
FIG. 13 is a photograph of a Acer truncatum with bud stem section of the present invention after 28d cultivation in an axillary bud induction medium;
FIGS. 14 and 15 are photographs of Acer truncatum with bud stem sections of the present invention after 35d cultivation in an axillary bud induction medium;
Detailed Description
The following examples are intended to further illustrate the invention, but not to limit it.
Example 1
The following operations are sequentially carried out:
1. explant disinfection treatment: in the morning of 2021 on sunny days, mature, full and pest-free Acer truncatum seeds are collected and brought back to a laboratory after water spraying. And (5) placing the collected Acer truncatum seeds in a refrigerator at the temperature of 4 ℃ for refrigeration, and taking out after 1 d. Adding a proper amount of washing powder, washing under tap water for 20-50 min, treating the inner and outer seed coats of the seeds (see figure 1) according to the method of table 1, continuing washing for 1h, and finally washing with sterile water for 3-5 times. Disinfection with 70% alcohol, 0.1% mercuric chloride was performed and different time gradients were set as in table 2. During the disinfection process, forceps are used for stirring continuously, sterile water is used for flushing for 5 to 8 times, and sterile filter paper is used for sucking the water on the surface.
TABLE 1 disinfecting effect of different seed treatments on Acer Truncatum Bunge seeds
TABLE 2 disinfecting effects of different disinfecting treatments on Acer Truncatum Bunge seeds
Finally, it was confirmed that the preferred sterilization method of the present invention is as follows:
(1) Treatment of explants: and (3) placing the collected mature, full and pest-free acer truncatum seeds in a refrigerator at the temperature of 4 ℃ for refrigeration, and taking out after 1 d. Adding a proper amount of washing powder, washing under tap water for 40min, peeling off exodermis, continuing washing for 1h, and finally washing with sterile water for 3 times.
(2) Explant sterilization: the explant washed with sterile water was placed in an ultra clean bench and soaked in 70% by volume alcohol for 45s and rinsed 3 times with sterile water. Soaking Acer truncatum seeds in 0.1% mercuric chloride solution for 8min, and repeatedly cleaning with sterile water for 5-8 times. Finally, the seeds are placed on sterile filter paper to absorb redundant moisture on the surfaces of the seeds.
2. After the sterilized Acer truncatum seeds are placed on sterile filter paper to absorb water, the inner seed coats are carefully selected by a surgical knife and forceps, the embryonal axis and the cotyledons are separated, the embryonal axis is inoculated into a culture medium according to the hormone formula shown in Table 3 for germination culture, and the germination rate can reach 95% at most under the illumination condition (see figure 2-7). Adding 30g/L of sucrose, 7g/L of agar and pH 5.5-5.8; the culture temperature is 24-2 ℃, the illumination intensity is 2100-2200lx, and the illumination time is 12-15h/d.
TABLE 3 Effect of different plant growth regulators on Acer Truncatum Bunge embryo germination culture
Finally, it was confirmed that the preferred germination medium of the present invention is: MS+1.5 mg/L2, 4-D+0.75mg/L PVP.
3. The root of the acer truncatum aseptic seedling obtained in the above step is cut off, and the acer truncatum aseptic seedling is inoculated into an MS culture medium directly inoculated with the hormone formula shown in Table 4 for subculture. The number of leaves and branches are greatly increased, and the rooting number can reach 6.00 at most (see figures 8-11). Culturing under light for 35d, adding 30g/L sucrose, 7g/L agar and pH 5.5. The culture temperature is 24-2 ℃, the illumination intensity is 2100-2200lx, and the illumination time is 12-15h/d.
Table 4 selection of sterile seedling subculture Medium for Acer truncatum seed embryo
Finally, the preferred subculture medium of the present invention was confirmed to be: MS+1.0mg/L IBA+0.75mg/L PVP.
4. And (3) placing the acer truncatum aseptic seedlings obtained in the steps in an ultra-clean workbench, cutting off leaves, and leaving long leaf stalks of 1 cm. Cutting the plant into stem segments with at least one axillary bud and 1-3 cm long. The stem sections were then inoculated directly into MS medium according to the hormone formulation shown in Table 5 for axillary bud induction. Culturing in dark for 3d, and culturing under illumination; after 35d of light culture, axillary bud induction rate was counted (see FIG. 12-15), 30g/L of sucrose was added, 7g/L of agar was added, and pH was 5.5. The culture temperature is 24-2 ℃, the illumination intensity is 2100-2200lx, and the illumination time is 12-15h/d.
TABLE 5 influence of different hormone ratios on Acer Truncatum Bunge bud stem axillary bud induction
Finally, it was confirmed that the preferred axillary bud induction medium of the present invention is: MS+0.75mg/L PVP+3.0 mg/L6-BA+0.1 mg/L IBA.
Claims (9)
1. A method for sterilizing an acer truncatum seed explant, comprising the steps of:
(1) Explant selection: mature, full and pest-free Acer truncatum seeds.
(2) Explant treatment: and (5) placing the collected Acer truncatum seeds in a refrigerator at the temperature of 4 ℃ for refrigeration, and taking out after 1 d. Adding a proper amount of washing powder, washing under tap water for 20-50 min, peeling off the outer seed coat, continuing washing for 1h, and washing with sterile water for 3-5 times.
(3) Explant sterilization: placing the explant washed by sterile water in an ultra-clean workbench, soaking the explant in 70% alcohol solution for 30-60 s, and washing 3 times by sterile water. Soaking Acer truncatum seeds in 0.1% mercuric chloride solution for 3-10 min (the specific time depends on the lignification degree of seed coats), and repeatedly cleaning with sterile water for 5-8 times. Finally, the seeds are placed on sterile filter paper to absorb redundant moisture on the surfaces of the seeds.
2. The method for rapidly obtaining the acer truncatum aseptic seedlings is characterized by comprising the following steps of: after the sterilized Acer truncatum seeds are placed on sterile filter paper to absorb water, the inner seed coats are carefully selected by using a surgical knife and forceps, the cotyledons and the embryo axes are separated, and the embryo axes of the zygotes are inoculated into a culture medium for germination culture.
3. A method for inducing rapid proliferation of a budded stem segment of acer truncatum, comprising the steps of:
(1) The acer truncatum aseptic seedling obtained by the process is placed in an ultra-clean workbench, leaves are cut off, and long leaves with the length of 1cm are left. Cutting the plant into stem segments with at least one axillary bud and 1-3 cm long.
(2) Axillary bud induction: inoculating stem segments of Acer truncatum with single buds into a culture medium for axillary bud induction culture. Culturing in dark for 3d, and culturing under illumination.
4. The method for obtaining aseptic seedlings of Acer truncatum seeds according to claim 2, wherein the cultivation is carried out under light conditions for 28-35 d after the dark treatment for 3 d.
5. The method for obtaining aseptic seedlings of acer truncatum seeds according to claim 2, wherein the culture medium used for germination of the seeds is: MS+0.5-2.0 mg/L2, 4-D.
6. The method for rapid propagation of acer truncatum bud stem according to claim 3, wherein the culture medium used for axillary bud induction in the step (2) is MS+1.0-5.0 mg/L6-BA+0.1 mg/LIBA.
7. The method for rapid propagation of acer truncatum bud stem according to claim 3, wherein the step (2) is carried out after dark treatment for 3d and then is carried out under the condition of illumination for 28-35 d.
8. The method according to claim 2 or 3, wherein the cultivation temperature is 24.2℃and the light cultivation intensity is 2100 to 2200lx and the light cultivation time is 12 to 15 hours/d.
9. The rapid propagation method of Acer truncatum according to claims 2 and 3, wherein the culture medium used in the method is added with 30g/L sucrose, 0.75mg/L PVP (polyvinylpyrrolidone) and 7g/L agar, and the pH is adjusted to 5.5-5.8.
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